Dengue virus is the most prevalent human arbovirus on earth, infecting an estimated 50-100 million individuals annually. Despite this public health burden, there are currently no licensed vaccines or therapeutics to treat dengue. The host type I interferon response is known to play a critical role in limiting replication and spread of dengue virus, and as such dengue has evolved countermeasures by which it blocks interferon signaling, but not production, in infected cells. The NLR (nucleotide binding domain and leucine-rich-repeat-containing or NOD-like receptor) family of proteins has been identified as initiators and regulators of innate immune responses to a wide range of pathogens including some viruses. NLRX1, a new member of the NLR family, has been shown to act as a negative regulator of MAVS-mediated antiviral signaling, and studies have indicated that knockdown of NLRX1 expression leads to increased interferon production in the presence of viral infection. To date, however, the role of NLRX1 in dengue virus infection has not been explored. Additionally, dengue virus research has been limited by the lack of suitable small animal models in which to study dengue pathogenesis and host immune responses to infection. Those that have been developed are limited in their ability to study human innate immune responses to infection. The goal of this proposal is to examine the role of NLRX1 in the human innate immune response to a variety of relevant dengue virus infections using both in vitro and in vivo systems. Aim 1 will examine the functional role of NLRX1 in dengue- infected human cells. In this aim we will knockdown NLRX1 in human monocytic cells to determine how NLRX1 modulates MAVS-mediated antiviral signaling during infection with dengue. This will also be examined in the context of receptor-mediated vs. antibody-enhanced infection. Aim 2 will establish a novel humanized mouse model of viscerotropic dengue virus infection in which to study human immune responses. We will generate Rag2-gC double knockout (DKO) mice humanized with human immune cells and human liver cells (AFC8-hu HSC/Hpe mice) in order to examine viscerotropic DENV disease and human immune responses to infection. Aim 3 will utilize this humanized mouse model of DENV disease to assess the role human NLRX1 plays in innate immune response to and disease progression of DENV infection. We will use shRNA knockdown of NLRX1 in human cells engrafted into AFC8-hu HSC/Hpe mice and compare DENV infection with that of mice with unaltered NLRX1 levels. Taken together the results of this proposal will provide critical information about innate immune responses to DENV infection and provide a novel small animal model in which to study DENV pathogenesis and human immune responses.